Inorganic pyrophosphate ion has a central role in the calcification of bones and teeth through its effect on the rate of formation and growth of calcium phosphate crystals. Elevated levels of pyrophosphate ion have been found in the plasma and the synovial fluid of patients with osteoarthritis, pseudogout, acromegaly and uremia. Inorganic pyrophosphatase, an enzyme which is found in all cellular life, controls the level of pyrophosphate ion. It is the object of this proposal to determine the high resolution X-ray crystal structure of inorganic pyrophosphatase from Bakers' yeast (PPase). PPase crystallizes in the space group P21 with one dimeric molecule of molecular weight 68,000 in the asymmetric unit of its unit cell. The diffraction pattern of the crystals exhibits significant presence out to at least a resolution corresponding to spacings of 2.5 Angstrom units. X-ray diffraction data will be collected on precession films and measured by a scanning microdensitometer. The phases of the reflections will be determined using the techniques of isomorphous replacement and anomalous dispersion. The high resolution three dimensional structure of the enzyme will be elucidated from consideration of the resulting electron density map together with the known amino acid sequence of the protein. The mode of substrate binding and any changes in protein conformation on binding the substrate will be determined from X-ray structural studies of enzyme-inhibitor complexes. This structural information, when taken together with chemical and kinetic data, should indicate the catalytic mechanism of PPase. This, in turn, should lead to a greater understanding of normal and pathological states of mineralized tissues. BIBLIOGRAPHIC REFERENCE: G. Bunick, G.P. McKenna, R. Colton and D. Voet, The X-ray Structure of Yeast Inorganic Pyrophosphatase. Crystal Properties, J. Biol. Chem. 249,4647(1974).